Neuromuscular diseases are characterized by impaired functioning of the muscles due to either muscle or nerve pathology (myopathies and neuropathies). The neuropathies are characterized by neurodegeneration and impaired nerve control leading to problems with movement, spasticity or paralysis. Examples include Huntington's disease (HD), several types of spinocerebellar ataxia (SCA), Friedreich's ataxia (FA), Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal dementia (FTD). A subset of neuropathies is caused by a cis-element repeat instability. For instance, HD is caused by a triplet (CAG)n repeat expansion in exon 1 of the HTT gene. Expansion of these repeats results in expansion of a glutamine stretch at the N-terminal end of the 348 kDa cytoplasmic huntingtin protein. Huntingtin has a characteristic sequence of 6 to 29 glutamine amino acid residues in the normal form; the mutated huntingtin causing the disease has more than 38 residues. The continuous expression of mutant huntingtin molecules in neuronal cells results in the formation of large protein deposits which eventually give rise to cell death, especially in the frontal lobes and the basal ganglia (mainly in the caudate nucleus). The severity of the disease is generally proportional to the number of extra residues. AONs specifically targeting the expanded CAG repeats (such as PS57 (CUG)7 as a 2′-O-methyl phosphorothioate RNA; SEQ ID NO:1 Evers et al.) can be applied to effectively reduce mutant huntingtin transcript and (toxic) protein levels in HD patient-derived cells. For treatment of neuropathies, systemically administered AONs need to pass the blood brain barrier. Thus, there is a need for optimization of oligochemistry allowing and/or exhibiting improved brain delivery.
The myopathies include genetic muscular dystrophies that are characterized by progressive weakness and degeneration of skeletal, heart and/or smooth muscle. Examples of myopathies are Duchenne muscular dystrophy (DMD), myotonic dystrophy type 1 (DM1), and myotonic dystrophy type 2 (DM2). DM1 and DM2 are both also caused by cis-element repeat instability; DM1 by a trinucleotide (CTG)n repeat expansion in the 3′ untranslated region of exon 15 in the DMPK gene, and DM2 by a tetranucleotide (CCTG)n repeat expansion in the DM2/ZNF9 gene. Also here, AONs specifically targeting the expanded repeats, such as PS58, (CAG)7, a 2′-O-methyl phosphorothioate RNA for DM1 (Mulders et al.), have been shown to efficiently induce the specific degradation of the (toxic) expanded repeat transcripts. In contrast to DMD where the gene defect is associated with increased permeability of the muscle fiber membranes for small compounds as AONs, for most other myopathies an enhanced AON distribution to and uptake by muscle tissue is essential to obtain a therapeutic effect. Thus, also here there is a need for optimization of oligochemistry allowing and/or exhibiting improved muscle delivery.
The particular characteristics of a chosen chemistry at least in part affect the delivery of an AON to the target transcript: administration route, biostability, biodistribution, intra-tissue distribution, and cellular uptake and trafficking. In addition, further optimization of oligonucleotide chemistry is conceived to enhance binding affinity and stability, enhance activity, improve safety, and/or to reduce cost of goods by reducing length or improving synthesis and/or purification procedures. Multiple chemical modifications have become generally and/or commercially available to the research community (such as 2′-O-methyl RNA and 5-substituted pyrimidines and 2,6-diaminopurines), whereas most others still present significant synthetic effort to obtain. Especially preliminary encouraging results have been obtained using 2′-O-methyl phosphorothioate RNA containing modifications on the pyrimidine and purine bases as identified herein.
In conclusion, to enhance the therapeutic applicability of AONs for treating human cis-element repeat instability associated genetic disorders as exemplified herein, there is a need for AONs with further improved characteristics.